Abstract High throughput screening assays for GPCR drug discovery are hampered by the complex instrumentation associated with current technologies, and the high risk of false-positives and off- target effects. The majority of currently available GPCR drug screening assays are end-point assays that rely on secondary reporters of GPCR signaling events after signaling has occurred. These assays are further constrained by the requirement of an exogenous reagent for the assay readout. In the Phase I of this SBIR proposal, we propose to develop a novel reagent-free, real- time platform technology for GPCR signaling. The basis for the proposed technology is the use of our novel fluorescent protein, LucY (for Lucigen Yellow), as a protein interaction reporter. We propose to develop a split-fluorescence reassembly assay following the principles of Bimolecular Fluorescence Complementation (BiFC). LucY is advantageous to other split- fluorophore systems, like those based on GFP, because the reversible nature of its fluorescence allows for both 'signal on' and 'signal off' screening. Our proposed assay requires neither secondary reagents nor complex instrumentation, and thus minimizes the occurrence of false positives. Use of this technology in drug screening will require minimal investments in instrumentation and reagents, which is rarely an option for this class of targets.